The overall goal of the proposed research is to develop and apply methods for characterizing large proteins and protein complexes with other macromolecules or small ligands. This is of high significance since protein interactions are key to many biological processes and may be entry points for therapeutic interventions to fight human diseases. The planned research will include NMR, computational and biochemical approaches. NMR methods will heavily bank on non-uniform sampling approaches, which have been pioneered by this P0-1 and have been shown to dramatically extend the resolution of multidimensional NMR experiments matching the resolution achievable with modern NMR hardware. Of particular interest will be the focus on weakly interacting protein complexes that are difficult to crystallize, do not exhibit well-resolved NMR spectra and exhibit severe line broadening when studied with standard approaches. Thus, the P0-1 will tackle a serious problem of structural biology that can only be solved with new NMR approaches. The proposed research contains five research components and three cores. Component 1 (Wagner) will develop experimental and computational approaches for structural studies of large proteins and protein complexes. Component 2 (Wagner) proposes development and application of combined computational and experimental approaches for protein interactions with small molecules and other macromolecules for situations where experimental constraints are sparse. Component 3 (Hoch) proposes to further develop and apply methods for non-uniform sampling of protein spectra and processing of the nonuniformly acquired data. Component 4 (Walsh) targets structures and interactions of modules of the non-ribosomal polypeptide synthetases EntF and EntB. The overall goal is to understand the mechanisms by which bacteria make unusual peptides, some of which have important pharmacological properties. Component 5 (Chou) is focused on the development of new DNA-based alignment media for measuring residual dipolar couplings and strategies for obtaining new restraints for defining protein complexes both in aqueous solution and detergent micelles. The research will be supported by three cores for Administration (Core A), NMR instrumentation (Core B) and Computation (Core C). This P0-1 has had and will have a wide impact on biological science in the region. It stimulated numerous collaborations with other biologists and resulting in many publications that benefited from the expertise developed in this grant.